This invention relates to relatively short peptides about 6-45 residues in length, which are naturally available in minute amounts in the venom of the cone snails or analogs to the naturally available peptides, and which include one or more bromo-tryptophan residues.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
Mollusks of the genus Conus produce a highly toxic venom that enables them to carry out their unique predatory lifestyle. Prey are immobilized by the venom that is injected by means of a highly specialized venom apparatus, a disposable hollow tooth that functions both in the manner of a harpoon and a hypodermic needle.
Few interactions between organisms are more striking than those between a venomous animal and its envenomated victim. Venom may be used as a primary weapon to capture prey or as a defense mechanism. These venoms disrupt essential organ systems in the envenomated animal, and many of these venoms contain molecules directed to receptors and ion channels of neuromuscular systems.
The predatory cone snails (Conus) have developed a unique biological strategy. Their venom contains relatively small peptides that are targeted to various neuromuscular receptors and may be equivalent in their pharmacological diversity to the alkaloids of plants or secondary metabolites of microorganisms. Many of these peptides are among the smallest nucleic acid-encoded translation products having defined conformations, and as such, they are somewhat unusual. Peptides in this size range normally equilibrate among many conformations. Proteins having a fixed conformation are generally much larger.
The cone snails that produce these toxic peptides, which are generally referred to as conotoxins or conotoxin peptides, are a large genus of venomous gastropods comprising approximately 500 species. All cone snail species are predators that inject venom to capture prey, and the spectrum of animals that the genus as a whole can envenomate is broad. A wide variety of hunting strategies are used, however, every Conus species uses fundamentally the same basic pattern of envenomation.
Several peptides isolated from Conus venoms have been characterized. These include the .alpha.-, .mu.- and .omega.-conotoxins which target nicotinic acetylcholine receptors, muscle sodium channels, and neuronal calcium channels, respectively (Olivera et al., 1985). Conopressins, which are vasopressin analogs, have also been identified (Cruz et al. 1987). In addition, peptides named conantokins have been isolated from Conus geographus and Conus tulipa (Mena et al., 1990; Haack et al., 1990). These peptides have unusual age-dependent physiological effects: they induce a sleep-like state in mice younger than two weeks and hyperactive behavior in mice older than 3 weeks (Haack et al., 1990). Recently, peptides named contryphans containing D-tryptophan residues have been isolated from Conus radiatus (U.S. Ser. No. 60/).
The N-methyl-D-aspartate (NMDA) receptor is a postsynaptic ionotropic receptor which is responsive to the excitatory amino acids glutamate and glycine and the synthetic compound NMDA, among others. The NMDA receptor controls the flow of both divalent (Ca.sup.2+) and monovalent (K.sup.+, Na.sup.+) ions into the postsynaptic neural cell through a receptor associated channel (Foster and Fagg, 1987; Mayer and Miller, 1990).
The NMDA receptor has been implicated during development in specifying neuronal architecture and synaptic connectivity, and may be involved in experience dependent synaptic modifications. In addition, NMDA receptors are also thought to be involved in long term potentiation, Central Nervous System (CNS) plasticity, cognitive processes, memory acquisition, retention and learning. The NMDA receptor has also drawn particular interest since it appears to be involved in a broad spectrum of CNS disorders. For example, during brain ischemia caused by stroke or traumatic injury, excessive amounts of the excitatory amino acid glutamate are released from damaged or oxygen deprived neurons. This excess glutamate binds to the NMDA receptor which opens the ligand-gated ion channel thereby allowing Ca.sup.++ influx producing a high level of intracellular Ca.sup.++ which activates biochemical cascades resulting in protein, DNA and membrane degradation leading to cell death. This phenomenon, known as excitotoxicity, is also thought to be responsible for the neurological damage associated with other disorders ranging from hypoglycemia and cardiac arrest to epilepsy. In addition, there are reports indicating similar involvement in the chronic neurodegeneration of Huntington's, Parkinson's and Alzheimer's diseases. Activation of the NMDA receptor has been shown to be responsible for post-stroke convulsions, and, in certain models of epilepsy, activation of the NMDA receptor has been shown to be necessary for the generation of seizures.
In addition to the effect of glutamate on the NMDA channel, it is also regulated by glycine. This amino acid increases NMDA-evoked currents in various tissues Johnson and Ascher, 1987; Kleckner and Dingledine, 1988! by increasing the opening frequency of the NMDA channel Johnson and Ascher, 1987!. Thus, NMDA-induced calcium influx and intracellular accumulation may therefor also be stimulated by glycine Reynolds et al., 1987; Wroblewski et al., 1989!, which interacts with its own distinct site Williams et al., 1991!.
Neuropsychiatric involvement of NMDA receptor has also been recognized. Blockage of the NMDA receptor Ca.sup.2+ channel by the animal anesthetic PCP (phencyclidine) produces a psychotic state in humans similar to schizophrenia (Johnson and Jones, 1990). NMDA receptors have also been implicated in certain types of spatial learning (Bliss et al., 1993). Interestingly, both the spatial and temporal distribution of NMDA receptors in mammalian nervous systems have been found to vary. Thus, cells may produce NMDA receptors at different times in their life cycles and not all neural cells may utilize the NMDA receptor.
Specifically, it has been shown that neurotransmission mediated through the NMDA receptor complex is associated with seizures Bowyer, 1982; McNamara et al., 1988!, ischemic neuronal injury Simon et al., 1984; Park et al., 1988! and other phenomena including synaptogenesis Cline et al., 1987!, spatial learning Morris et al., 1986! and long-term potentiation Collinridge et al., 1983; Harris et al., 1984; Morris et al., 1986!. Regulation of these neuronal mechanisms by NMDA-mediated processes may involve activation of a receptor-gated calcium ion channel Nowak et al., 1984; Mayer et al., 1987; Ascher and Nowak, 1988!.
Epilepsy is a recurrent paroxysmal disorder of cerebral function characterized by sudden brief attacks of altered consciousness, motor activity, sensory phenomena or inappropriate behavior caused by abnormal excessive discharge of cerebral neurons. Convulsive seizures, the most common form of attacks, begin with loss of consciousness and motor control, and tonic or clonic jerking of all extremities but any recurrent seizure pattern may be termed epilepsy. The term primary or idiopathic epilepsy denotes those cases where no cause for the seizures can be identified. Secondary or symptomatic epilepsy designates the disorder when it is associated with such factors as trauma, neoplasm, infection, developmental abnormalities, cerebrovascular disease, or various metabolic conditions. Epileptic seizures are classified as partial seizures (focal, local seizures) or generalized seizures (convulsive or nonconvulsive). Classes of partial seizures include simple partial seizures, complex partial seizures and partial seizures secondarily generalized. Classes of generalized seizures include absence seizures, atypical absence seizures, myoclonic seizures, clonic seizures, tonic seizures, tonic-clonic seizures (grand mal) and atonic seizures. Therapeutics having anticonvulsant properties are used in the treatment of seizures. Most therapeutics used to abolish or attenuate seizures demonstrate act at least through effects that reduce the spread of excitation from seizure foci and prevent detonation and disruption of function of normal aggregates of neurons. Anticonvulsants which have been utilized include phenytoin, phenobarbital, primidone, carbamazepine, ethosuximide, clonazepam and valproate. For further details of seizures and their therapy (see Rall & Schleifer (1985) and The Merck Manual (1992)).
The development of drugs acting at the NMDA receptor is described in U.S. Pat. Nos. 4,904,681; 5,061,721 and 5,086,072 and in PCT application publication numbers WO 94/07914 and WO 96/11698. It is desired to identify additional compounds which target the NMDA receptor. It is further desired to identify compounds which are useful as anticonvulsant or neuroprotective agents.